How to: Isolation of primary Neutrophils

Neutrophils are a major component of the immune system and can account for 60–70% of leukocytes in humans. Because there isn’t a well-established, long-term neutrophil cell line that can be used in research, one must isolate them from blood to perform functional studies. Like monocytes, extracting these immune cells from patients can provide valuable insights.

Reagents

• Apheresis leukoreduction collar or whole blood
• PBS
• Ficoll Paque Plus (GE Healthcare, #GE17–1440–02)
• 0.2% NaCl solution (1 g NaCl + 500 ml Millipore water)
• 1.6% NaCl solution (8 g NaCl + 500 ml Millipore water)
• RPMI 1640
• Human serum albumin
• TC-supplements (10 U/ml Penicillin G, 100 ug/ml Streptomycin, 6 mM Acid-free HEPES, 1.6 mM L-glutamine, 50 uM beta-mercaptoethanol)

All vendors are suggestions and are provided to look up manufacturer protocols.

Protocol

1. Prepare NaCl stock solutions: fully dissolve NaCl in water and filter the solutions in a cell culture hood to keep them sterile.
2. Drain the collar (approx. 10 ml) into a 50 ml tube when using a leukoreduction collar. Dilute it 1:3 with PBS to reinstate the volume of whole blood (approx. 30 ml). This step can be skipped if using whole blood.
3. Layer the blood on top of 15 ml Ficoll. Therefore fill 15 ml Ficoll into a 50 ml tube, tilt the falcon until the liquid surface is almost horizontal, and then slowly let the blood flow along the wall of the falcon. Due to its lower density, the blood will layer on top of the Ficoll. Use a stripette for this step and set the pipetboy to a minimum speed. Be careful to not disturb the phase separation between Ficoll and blood when moving the falcon or tilting it back vertically.
4. Centrifuge for 1500 rpm for 25 minutes with break OFF!
5. After centrifugation, you will see a red pellet (containing our neutrophils, together with the erythrocytes), a Ficoll-paque, a PBMC (peripheral blood mononuclear cells)-ring, and the plasma on top. Remove everything except the red blood cell (RBC) pellet.
6. Add 20 ml of the 0.1% NaCl solution to the RBC pellet. Resuspend the pellet by inverting for 15 seconds. Then add 20 ml of the 1.6% NaCl solution to re-establish osmolarity. This step causes the more fragile erythrocytes to “explode”, thus removing them from the more robust neutrophils.
7. Centrifuge for 15 minutes at 200 g with break ON!
8. Aspirate the supernatant and be careful to not disturb the pellet.
9. Repeat steps 6.-8. until the pellet is white and not red anymore. All erythrocytes should be removed now.
10. Resuspend the neutrophils in RPMI containing 10% human serum and 1% penicillin-streptomycin. Primary neutrophils can e.g. be seeded at 2x10⁶ cells/well in a 48-well plate.

Caution: All samples should be processed in an appropriate BSL cell culture hood.

Tips

• Alternatively, neutrophils can be purified by negative selection using e.g. the EasySep direct human neutrophil isolation cocktail before performing the Ficoll density centrifugation. This kit labels all other cells besides the neutrophils which can make sense if you e.g. want to stain CD15 or CD16 with an antibody or want to do functional studies on this receptor afterward.